Rapid and Quantitative Intraoperative Pathology-Assisted Surgery by Paired-Agent Imaging-Derived Confidence Map

Purpose In nonmetastatic head and neck cancer treatment, surgical margin status is the most important prognosticator of recurrence and patient survival. Fresh frozen sectioning (FFS) of tissue margins is the standard of care for intraoperative margin assessment. However, FFS is time intensive, and its accuracy is not consistent among institutes. Mapping the epidermal growth factor receptor (EGFR) using paired-agent imaging (PAI) has the potential to provide more consistent intraoperative margin assessment in a fraction of the time as FFS. Procedures PAI was carried out through IV injection of an anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) and an untargeted IRDye680LT carboxylate. Imaging was performed on 4 µm frozen sections from three oral squamous cell carcinoma xenograft mouse models ( n = 24, 8 samples per cell line). The diagnostic ability and tumor contrast were compared between binding potential, targeted, and untargeted images. Confidence maps were constructed based on group histogram-derived tumor probability curves. Tumor differentiability and contrast by confidence maps were evaluated. Results PAI outperformed ABY-029 and IRDye 680LT alone, demonstrating the highest individual receiver operating characteristic (ROC) curve area under the curve (PAI AUC: 0.91, 0.90, and 0.79) and contrast-to-noise ratio (PAI CNR: 1, 1.1, and 0.6) for FaDu, Det 562, and A253. PAI confidence maps (PAI CM) maintain high tumor diagnostic ability (PAI CMAUC: 0.91, 0.90, and 0.79) while significantly enhancing tumor contrast (PAI CMCNR: 1.5, 1.3, and 0.8) in FaDu, Det 562, and A253. Additionally, the PAI confidence map allows avascular A253 to be differentiated from a healthy tissue with significantly higher contrast than PAI. Notably, PAI does not require additional staining and therefore significantly reduces the tumor delineation time in a 5 × 5 mm slice from 35 min to under a minute. Conclusion This study demonstrated that PAI improved tumor detection in frozen sections with high diagnostic accuracy and rapid analysis times. The novel PAI confidence map improved the contrast in vascular tumors and differentiability in avascular tumors. With a larger database, the PAI confidence map promises to standardize fluorescence imaging in intraoperative pathology-assisted surgery (IPAS).